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Involvement of pallidal and nigral gaba mechanisms in the generation of tremulous jaw movements in rats
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Neuroscience Vol. 80, No. 2, pp. 535–544, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(97)00087-0
INVOLVEMENT OF PALLIDAL AND NIGRAL GABA MECHANISMS IN THE GENERATION OF TREMULOUS JAW MOVEMENTS IN RATS M. FINN, A. J. MAYORGA, A. CONLAN and J. D. SALAMONE* Department of Psychology, University of Connecticut, Storrs, CT 06269-1020, U.S.A. Abstract––Four experiments were conducted to investigate the role of pallidal and nigral GABA in the generation of tremulous jaw movements in rats. In these experiments, tremulous jaw movements were induced by i.p. injections of the anticholinesterase tacrine (5.0 mg/kg). Previous work has shown that the tremulous jaw movements induced by cholinomimetics and dopamine depletion are dependent upon striatal mechanisms. Thus, the present study investigated potential striatal output pathways that could be involved in the generation of these movements. Because there are GABAergic projections from neostriatum to entopeduncular nucleus (medial globus pallidus) and substantia nigra pars reticulata, the GABA agonist muscimol was injected directly into these structures to study the effects of GABA stimulation on tacrine-induced jaw movements. Injections of muscimol into the entopeduncular nucleus (25–100 ng) failed to have any significant effects on tacrine-induced vacuous jaw movements. However, injections of muscimol (12.5–50 ng) into the substantia nigra pars reticulata blocked the jaw movements induced by tacrine. In the third experiment, it was again demonstrated that 25.0 ng of muscimol injected directly into the substantia nigra pars reticulata blocked the jaw movements induced by tacrine; in addition, it was shown that injections of this dose 2.0 mm dorsal to the substantia nigra pars reticulata failed to affect tacrine-induced tremulous jaw movements. It was shown in the fourth experiment that injections of muscimol into a more medial portion of the substantia nigra pars reticulata also reduced tacrine-induced tremulous jaw movements. These results indicate that stimulation of GABAA receptors in substantia nigra pars reticulata can block tacrine-induced tremulous jaw movements. This finding is consistent with the notion that striatonigral GABA projections are involved in the generation of tremulous jaw movements. It is also possible that striatonigral GABA mechanisms are involved in human clinical phenomena such as parkinsonian tremor. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: acetylcholine, tremor, Parkinsonism, substantia nigra, globus pallidus, motor, striatum, dopamine.
Drug-induced perioral movements in rats have frequently been used to study extrapyramidal motor function.15,41,43 One of the most widely investigated of these perioral movements is vacuous or tremulous jaw movements (also known as vacuous or purposeless chewing). Vacuous jaw movements are defined as vertical deflections of the lower jaw that resemble chewing but are not directed towards any particular stimuli (e.g., Refs 44 and 45). These movements are induced by dopamine antagonists,41,46,47 reserpine,4,43,47 and neurotoxic depletions of dopamine in the ventrolateral portion of the neostriatum.18,27 Stimulation of muscarinic acetylcholine receptors also leads to vacuous jaw movements.5,9,41,44,45,49 As was the case with dopamine depletions, striatal mechanisms appear to be critical for the generation of cholinomimetic-induced vacuous jaw movements. Kelley et al.29 injected physostigmine into several striatal subregions, and reported that the most effective site was the ventrolateral striatum. Salamone *To whom correspondence should be addressed. Abbreviations: SNr, substantia nigra pars reticulata.
et al.45 observed that the ventrolateral striatum was the most effective site for the induction of jaw movements by the muscarinic agonist pilocarpine. In a recent study,34 it was reported that the anticholinesterase tacrine induced vacuous jaw movements in the range of 2.5–10.0 mg/kg. The jaw movements induced by 5.0 mg/kg tacrine were completely blocked by injection of 2.5–10 µg scopolamine into the ventrolateral striatum.34 Although the clinical significance of vacuous jaw movements remains uncertain (see Refs 15 and 42), it has been suggested that vacuous jaw movements could represent a rat model of parkinsonian tremor.18,27,34,45 Using a slow-motion videotape method, it has been shown that the jaw movements induced by reserpine,43 striatal dopamine depletion,18 pilocarpine18 and tacrine34 occur as repetitive bursts of movement with a peak in the 3.0–6.6 Hz frequency range. Electromyographic analyses of jaw muscles indicate that the temporalis muscle shows rhythmic bursts of activity that correspond to the tacrine-induced jaw movements.11 The local frequency of the jaw movements induced by dopamine
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depletion and cholinomimetics is consistent with the parkinsonian resting tremor frequency,1,26 but is not consistent with the 1–2 Hz frequency typically reported for tardive dyskinesia.54 Thus, it has been noted that these jaw movements are not only vacuous (i.e. non-directed), but are also tremulous (i.e. characterized by a periodic oscillation of a body member, see Refs 16 and 17). The acetylcholine–dopamine interaction that occurs in tremulous jaw movements is similar to that shown in human Parkinsonism. For example, tremulous jaw movements are induced by striatal dopamine depletions and cholinomimetic drugs; these conditions are also associated with human Parkinsonism.3,14,23,35,36 Tremulous jaw movements induced by reserpine can be reduced by the anticholinergic scopolamine,43 and it has been shown that anticholinergic drugs reduce parkinsonian symptoms but exacerbate tardive dyskinesia.7,14 The jaw movements induced by pilocarpine or reserpine can be reduced by moderate doses of the dopamine agonist apomorphine,4,48 and more recently it was shown that the jaw movements induced by the anticholinesterase tacrine can be reduced by various antiparkinsonian drugs, including apomorphine, bromocriptine, amantadine, L-DOPA and benztropine.12 Thus, it is possible that further research into tremulous jaw movements could be useful for helping to characterize the neurochemical and anatomical basis of extrapyramidal motor functions, particularly those mechanisms related to Parkinsonism. One of the most important questions remaining in this area concerns the anatomical pathway that is necessary for generating tremulous jaw movements. As noted above, striatal dopamine and acetylcholine mechanisms are critical for the generation of these movements. Yet the striatal output pathway through which jaw movements are stimulated remains uncertain. The two major striatofugal pathways project either to the medial segment of the globus pallidus (or entopeduncular nucleus in non-primates) or the substantia nigra pars reticulata (SNr).19,20,32,37,38 The medial globus pallidus and SNr are both considered to be major output nuclei of the basal ganglia.39,55 The direct striatofugal pathways utilize GABA as a neurotransmitter,21,52,55 and stimulation of GABA receptors in the SNr has been shown to produce motor effects such as circling behaviour.53 Thus, it is possible that manipulation of GABA transmission in the entopeduncular nucleus or SNr could affect the neural circuitry that generates tremulous jaw movements. Four experiments were conducted to study the role of pallidal and nigral GABA in the production of tremulous jaw movements. In the first two experiments, microinjections of the GABAA agonist muscimol into the entopeduncular nucleus and the lateral SNr of the rat were studied for their effects upon tacrine-induced tremulous jaw movements. Based upon the results of the first two experiments, a third experiment was conducted in which the effects of
intra-nigral muscimol were replicated, and muscimol was also injected into a site dorsal to the SNr in order to control for possible spread of the drug from the SNr injection site. In the fourth experiment, muscimol was injected into a more medial SNr injection site. EXPERIMENTAL PROCEDURES
Subjects The subjects were 113 male Sprague–Dawley rats obtained from Harlan Sprague–Dawley (Indianapolis, IN). All rats were housed individually in a colony room maintained at 23)C with a 12 h light/dark cycle (lights on at 07.00). Standard lab chow and water were available ad libitum and all behavioural testing was performed 4–9 h after light onset. University guidelines on animal care and experimentation were followed for these studies. Drugs All drugs were obtained from Sigma Chemical Co. Both tacrine and muscimol were dissolved in 0.9% saline. Tacrine was administered i.p. immediately following the intracranial injection of muscimol or saline. Behavioural testing was initiated 10 min after the injection of tacrine. Behavioural observation procedures The observation chamber consisted of a clear Plexiglas box measuring 28#28#28 cm3, which had a wire mesh floor. The box was elevated 42 cm from the surface of the table, allowing behavioural observation from all angles. An observer who was unaware of the drug treatment recorded vacuous jaw movements for a 5 min period using a mechanical counter. Vacuous jaw movements were defined as vertical deflections of the lower jaw that resembled chewing but were not directed at any particular stimulus. Yawning, gaping and tongue protrusions were not counted as vacuous jaw movements. Each individual deflection of the jaw was counted as a jaw movement. Previous research has shown a high degree of interrater reliability (i.e. correlations between two observers greater than r=+0.9) with the use of these observational methods for recording jaw movements (see Ref. 34). Surgery Rats were anaesthetized with sodium pentobarbital (50.0 mg/kg). Stainless steel guide cannulae were implanted using standard stereotaxic procedures. The stereotaxic coordinates for the entopeduncular nucleus were the following (in mm, with incisor bar 5.0 mm above interaural line, see Ref. 40): A-P, "0.6 from bregma, M-L, &2.7 from midline, D-V, "6.4 from skull. For the lateral SNr site, the coordinates were A-P, "3.0, M-L, &2.8, D-V, "7.0. The coordinates for the dorsal control site were A-P, "3.0, M-L, &2.8, D-V, "5.0. The coordinates for the middle SNr site were: A-P, "3.0, M-L, &1.8, D-V, "7.2. The cannulae were anchored to the skull with stainless steel screws and dental cement. Wire stylets were placed into the guides to prevent occlusion. A recovery period of seven days was allowed before experimental testing. Intracranial drug injections Injections were made with 30-gauge injection cannulae extending 2.0 mm below the guide cannulae for the SNr (D-V, "9.0 from the skull for lateral SNr, D-V, "9.2 for middle SNr) and the dorsal control site (D-V, "7.0), and 1.0 mm below the guide cannulae for the entopeduncular nucleus (D-V, "7.4). The injectors were attached via PE-10 tubing to a 10 µl Hamilton syringe driven by a syringe pump
Nigral GABA and jaw movements (Harvard Apparatus). Bilateral microinfusions were made in a total volume of 1.0 µl, at a rate of 0.5 µl/min for each side. The injection cannulae were left in place for 1 min following drug infusion to allow for diffusion.
Table 1. The effect of muscimol injected into entopeduncular nucleus on tacrine-induced tremulous jaw movements in experiment 1 Drug treatment
Experiments In the first experiment rats were injected with either saline (n=7), 25.0 (n=7), 50.0 (n=6) or 100.0 ng (n=6) of muscimol per side into the entopeduncular nucleus, followed by 5.0 mg/kg of tacrine i.p. In the second experiment, 29 rats received intracranial injections of either saline (n=8), 12.5 (n=7), 25.0 (n=7) or 50.0 ng (n=7) of muscimol per side into the lateral SNr, followed by 5.0 mg/kg of tacrine i.p. In the third experiment, three groups were compared. One group (n=8) had cannulae implanted into lateral SNr, as described above, and received injections of 1.0 ul saline vehicle through the cannulae as well as 5.0 mg/kg tacrine. A second group (n=9) also had cannulae implanted into lateral SNr, and received tacrine plus 25.0 ng muscimol per side. In the third group, rats (n=12) were implanted with an injection target site 2.0 mm dorsal to the SNr (dorsal control site, see above). These rats received 25.0 ng of muscimol per side through the cannulae followed by 5.0 mg/kg of tacrine i.p. In the fourth experiment, rats were implanted with cannulae in the middle SNr as described above, and received intracranial injections of saline (n=8), 6.25 (n=6), 12.5 (n=7) or 25.0 ng (n=8) of muscimol per side, followed by 5.0 mg/kg of tacrine i.p. All animals were observed as described above for a 5 min period that was initiated 10 min after the tacrine injection. Histology Upon completion of the experiment, rats were deeply anaesthetized with sodium pentobarbital and perfused transcardially with physiological saline followed by 10% formalin to fix the brains. The brains were then removed and placed in 10% formalin for at least two days. They were cut into 75 µm coronal sections, mounted on glass slides and stained with Cresyl Violet to determine the location of the cannulae tracks. Data analysis Jaw movement data in each experiment were analysed by simple analysis of variance (ANOVA; Systat 5.0). Each dose of muscimol was compared with the effects of saline vehicle injection by using non-orthogonal planned comparisons that employed the overall error term from the ANOVA.30 RESULTS
Table 1 shows the results of the first experiment, in which muscimol was injected into the entopeduncular nucleus. There was no significant effect of muscimol on the jaw movements induced by 5.0 mg/kg tacrine (F3,22=0.637, P=0.599). The effects of SNr injections of muscimol on tremulous jaw movements induced by 5.0 mg/kg tacrine (the second experiment) are shown in Table 2. As shown in this table, there was a significant reduction in tremulous jaw movements produced by injection of muscimol (F3,25=5.966, P<0.01). Planned comparisons indicated that 25.0 ng and 50.0 ng doses of muscimol significantly reduced tremulous jaw movements relative to tacrine alone. In Table 3, the results of the third experiment are shown. There was an overall effect of drug treatment on tremulous jaw movements (F2,26=5.95, P<0.01). Planned comparisons revealed that injections of 25.0 ng muscimol into the SNr significantly reduced
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5.0 mg/kg tacrine+vehicle 5.0 mg/kg tacrine+25.0 ng/ side muscimol 5.0 mg/kg tacrine+50.0 ng/ side muscimol 5.0 mg/kg tacrine+100 ng/ side muscimol
Number of jaw movements Mean 168.0
S.E.M. 25.7
135.3
23.0
112.3
34.5
131.0
34.0
All rats received 5.0 mg/kg tacrine, as well as injections of vehicle or muscimol into the entopeduncular nucleus. Mean (&S.E.M.) number of jaw movements per 5 min observation period is shown for each treatment condition. Table 2. The effect of muscimol injected into substantia nigra pars reticulata on tacrine-induced tremulous jaw movements in experiment 2 Drug treatment 5.0 mg/kg tacrine+vehicle 5.0 mg/kg tacrine+12.5 ng/ side muscimol 5.0 mg/kg tacrine+25.0 ng/ side muscimol 5.0 mg/kg tacrine+50.0 ng/ side muscimol
Number of jaw movements Mean 125.1
S.E.M. 24.5
79.1
21.4
26.0*
13.4
29.1*
14.0
*P<0.05, different from tacrine plus vehicle. All rats received 5.0 mg/kg tacrine, as well as injections of vehicle or muscimol into the substantia nigra pars reticulata. Mean (&S.E.M.) number of jaw movements per 5 min observation period is shown for each treatment condition. Table 3. The results of experiment 3 Drug treatment
Mean
S.E.M.
5.0 mg/kg tacrine+vehicle/SNr 5.0 mg/kg tacrine+25.0 ng muscimol/SNr 5.0 mg/kg tacrine+25.0 ng muscimol/dorsal
102.8
19.9
33.0* 122.8
10.6 17.3
*P<0.05, different from tacrine plus vehicle. All rats received 5.0 mg/kg tacrine, as well as injections of either vehicle into substantia nigra pars reticulata (SNr), 25.0 ng muscimol into the SNr or 25.0 ng muscimol into the dorsal control site. Mean (&S.E.M.) number of jaw movements per 5 min observation period is shown for each treatment condition.
tremulous jaw movements relative to tacrine alone; this finding replicates the results of the previous study. However, injections of 25.0 ng muscimol 2.0 mm dorsal to SNr into overlying brain areas had no significant effect on tremulous jaw movements. The effects of muscimol injected into the middle SNr are shown in Table 4. There was a significant overall
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Table 4. The effect of muscimol injected into the middle substantia nigra pars reticulata site on tacrine-induced tremulous jaw movements in experiment 4 Drug treatment
Number of jaw movements
5.0 mg/kg tacrine+vehicle 5.0 mg/kg tacrine+6.25 ng/ side muscimol 5.0 mg/kg tacrine+12.5 ng/ side muscimol 5.0 mg/kg tacrine+25.0 ng/ side muscimol
Mean 87.8
S.E.M. 16.9
17.3*
9.3
11.4*
7.3
2.8*
1.8
*P<0.05, different from tacrine plus vehicle. All rats received 5.0 mg/kg tacrine, as well as injections of vehicle or muscimol into the substantia nigra pars reticulata. Mean (&S.E.M.) number of jaw movements per 5 min observation period is shown for each treatment condition.
effect of muscimol on tacrine-induced tremulous jaw movements (F3,25=13.2, P<0.001). Planned comparisons revealed that all three doses of muscimol plus tacrine significantly differed from tacrine alone. All cannula placements were verified by histological analyses. For simplicity, only those animals that received 25.0 ng muscimol in each of the four experiments have their placements shown in Figs 1–3 (left hemisphere only). Analysis of the histology demonstrated that cannulae were placed in the entopeduncular nucleus in the first experiment (Fig. 1), and in the anterolateral SNr in the second experiment (Fig. 2). The results of the histological analyses of the third and fourth experiments are shown in Fig. 3. For the group of rats that received injections of muscimol into the SNr, it can be seen that the placements are in the vicinity of the anterolateral SNr, as in the previous experiment. Rats in the dorsal control group had placements in the area of the zona incerta, posterior thalamus, lateral geniculate and reticular formation. One of the placements in this group was near the substantia nigra pars compacta, but none of them were in the subthalamic nucleus. The middle SNr placement (experiment 4) resulted in injections sites that were centrally located within the anterior SNr region. DISCUSSION
Stimulation of GABAA receptors in SNr by local injections of muscimol produced a dose-related reduction in tacrine-induced tremulous jaw movements. With placements in the lateral SNr, this effect was significant with injections of 25.0 ng muscimol, and some animals that received 25.0 or 50.0 ng muscimol showed a complete blockade of tacrineinduced tremulous jaw movements. The middle SNr injection site resulted in a more potent effect of muscimol, with significant effects at 6.25 and 12.5 ng doses. The effects of muscimol on tremulous jaw
movements were anatomically specific. There were no significant effects of muscimol injections into entopeduncular nucleus, even if doses as high as 100.0 ng were used. Moreover, injections of 25.0 ng muscimol 2.0 mm dorsal to SNr also failed to reduce tremulous jaw movements. The latter results suggest that muscimol injected into SNr is not reducing tremulous jaw movements simply because the drug is diffusing along the guide cannulae to other, more distant brain regions. Although the substantia nigra pars compacta is immediately dorsal to the SNr, it seems unlikely that muscimol is acting in this region to reduce tremulous jaw movements. GABAergic inhibition of substantia nigra dopamine-containing cells should, if anything, increase tremulous jaw movements. Previous work has shown that tremulous jaw movements are induced by dopamine antagonism or striatal dopamine depletion.4,18,27,46,47 Injections of low, presynaptically active doses of apomorphine increase, rather than decrease, reserpine-induced jaw movements.4 Tacrine-induced jaw movements are not blocked by doses of haloperidol up to 1.0 mg/ kg,51 so it is unlikely that inhibition of dopamine cells would block these movements. In addition, some of the rats that received implantations targeted at the SNr had placements that were quite ventral to the substantia nigra pars compacta; these rats still showed a blockade of tacrine-induced jaw movements. Thus, the present evidence, as well as previous work, suggests that stimulation of GABAA receptors in SNr reduces tremulous jaw movements. Anatomical evidence indicates that the medial globus pallidus/ entopeduncular nucleus and SNr are the two major output nuclei for both the ‘‘direct’’ and ‘‘indirect’’ pathways of striatal output.2,13,39 The former pathway represents a direct GABAergic projection from medium spiny cells in the neostriatum to the globus pallidus or SNr, while the latter involves connections through lateral globus pallidus and subthalamic nucleus. The present study demonstrates that the entopeduncular nucleus and SNr in the rat are not functionally equivalent in terms of tremulous jaw movements (see also Ref. 6). Rather, tremulous jaw movements seem to be generated through a mechanism that involves SNr, but not entopeduncular nucleus. Given current models of basal ganglia function, it is not clear why large doses of muscimol in the entopeduncular nucleus are ineffective for the suppression of tremulous jaw movements. It is possible that there are species differences in terms of the relative importance of medial globus pallidus and SNr as striatal output areas. Also, it should be mentioned that parkinsonian tremor in primates usually involves the hand; although jaw tremors do occur,1,25,50 they are less common than hand tremors. In rats, although we occasionally observe paw tremors, it is the jaw movements that are most obvious after dopamine depletions and cholinergic stimulation. It is possible that there are differences between the pathways through which forelimb and orofacial
Nigral GABA and jaw movements
Fig. 1. Cannulae placements for the first experiment are shown here. Placements are depicted for rats that received injections of 25.0 ng muscimol into the entopeduncular nucleus (black circles). EP, entopeduncular nucleus; see also Ref. 40.
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Fig. 2. Cannulae placements for the second experiment are shown here. Placements are shown for rats that received injections of 25.0 ng muscimol into the substantia nigra pars reticulata (black squares). SN, substantia nigra; see also Ref. 40.
Nigral GABA and jaw movements
Fig. 3. Placements for the third and fourth experiments are shown in this figure; placements are depicted for rats that received injections of 25.0 ng muscimol into the SNr (large black squares) and the dorsal control site (black circles) in experiment 3, as well as placements in the middle SNr site (triangles) in experiment 4. SN, substantia nigra; ZI, zona incerta; GL, lateral geniculate; see also Ref. 40.
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movements are influenced by striatal output. In rats, and possibly in other species as well, the SNr may be more important than the medial globus pallidus/ entopeduncular nucleus for conveying information that is necessary for oral motor function. It has been demonstrated that circuits connecting neostriatum, substantia nigra and superior colliculus are important for the generation of eye movements;10,28 future research will be necessary to determine the pathway through which cholinomimetic-stimulated jaw movements are generated. CONCLUSIONS
It has been suggested that neurons in pallidal output regions are hyperactive under the conditions that lead to Parkinsonism, and there is evidence to support this contention from parkinsonian patients.26 In fact, the presence of pallidal hyperactivity has been used as a rationale for using pallidotomies as a treatment for Parkinsonism.26,32 In the present study, it was observed that muscimol injected into SNr was very potent at reversing cholinomimetic-induced tremulous movements. This finding is consistent with the notion that neurons in the SNr are hyperactive during tremulous jaw movements, and that GABA-induced inhibition of SNr reverses both the neuronal hyperactivity and the jaw movements. The precise reason why conditions that lead to Parkinsonism would cause hyperactivity in
SNr neurons is not certain, although models of basal ganglia function have been proposed to explain this type of effect (e.g., Refs. 13 and 55). It is possible that interference with dopamine or stimulation of acetylcholine in the striatum leads to an inhibition of GABAergic neurons that project to SNr via the ‘‘direct’’ pathway.55 Also, it is possible that, through the connections with the lateral globus pallidus and subthalamic nucleus, parkinsonian conditions lead to an excess of glutamate-mediated excitation of SNr.6,22,31 Future research will be necessary to determine if either decreased GABA release or increased glutamate release in SNr is associated with the production of tremulous jaw movements in rats. Additional studies will be undertaken to identify other pharmacological conditions (e.g., SNr injections of GABA antagonists, excitatory amino acid agonists) that could lead to the production of tremulous jaw movements. Whether it is through glutamate or GABA mechanisms, or some combination of the two, the hyperexcitability of cells in the SNr could be a critical trigger for the generation of tremulous motor activity. Through connections with the thalamus or brainstem motor areas,52 the SNr could be instigating the oscillatory neural activity that ultimately manifests itself as tremulous movements.8 Acknowledgements—Many thanks to Bill Lexton, for his assistance with animal care. This work was supported by a grant from NIH (NINDS).
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(1984) Preliminary report: intracranial cholinergic drug infusion in patients with Alzheimer’s disease. Neurosurgery 15, 514–518. Hedreen J. C. and DeLong M. R. (1990) Organization of striatopallidal, striatonigral and nigrostriatal projections in the Macaque. J. comp. Neurol. 304, 569–595. Hunker C. J. and Abbs J. H. (1990) Uniform frequency of parkinsonian resting tremor in the lips, jaw, tongue and index finger. Movement Disord. 5, 71–77. Hutchison W. D., Lozano A. M., Davis K. D., Saint-Cyr J. A., Lang A. E. and Dostrovsky J. O. (1994) Differential neuronal activity in segments of globus pallidus in Parkinson’s disease patients. NeuroReport 21, 1533–1537. Jicha G. and Salamone J. D. (1991) Vacuous jaw movements and feeding deficits in rats with ventrolateral striatal dopamine depletions: possible model of parkinsonian symptoms. J. Neurosci. 11, 3822–3829. Kato M., Miyashita N., Hikosaka O., Matsumura M., Usui S. and Kori A. (1995) Eye movements in monkeys with local dopamine depletion in the caudate nucleus. I. Deficits in spontaneous saccades. J. Neurosci. 15, 912–927. Kelley A. E., Bakshi V. P., Delfs J. M. and Lang C. G. (1989) Cholinergic stimulation of the ventrolateral striatum elicits mouth movements in rats: pharmacological and regional specificity. Psychopharmacology 99, 542–549. Keppel G. (1982) Design and Analysis: A Researchers Handbook. Prentice–Hall, Englewood Cliffs, NJ. Klockgether T., Turski L., Honore T., Zhang Z., Gash D., Kurlan R. and Greenamyre J. T. (1991) The AMPA receptor antagonist NBXQ has antiparkinsonian effects in monoamine-depleted rats and MPTP-treated monkeys. Ann. Neurol. 30, 717–723. Loopjuit L. D. and Van Der Kooy D. (1985) Organization of the striatum: collateralization of its efferent axons. Brain Res. 348, 86–99. Lozano A. M., Lang A. E., Galvez-Jimenez N., Miyasaki J., Hutchison W. D. and Dostrovsky J. O. (1995) Effect of GPi pallidotomy on motor function in Parkinson’s disease. The Lancet 346, 1383-1387. Mayorga A. J., Carriero D. L., Cousins M. S., Gianutsos G. and Salamone J. D. (1997) Tremulous jaw movements produced by acute tacrine administration: possible relation to parkinsonian side effects. Pharmac. Biochem. Behav. 56, 273–279. Noring U., Povlesen U. J., Casey D. E. and Gerlach J. (1984) Effect of a cholinomimetic drug (RS 86) in tardive dyskinesia and drug-related Parkinsonism. Psychopharmacology 84, 569–571. Ott B. R. and Lannon M. C. (1992) Exacerbation of Parkinsonism by tacrine. Clin. Neuropharmac. 15, 322–325. Parent A., Bouchard C. and Smith Y. (1984) The striatopallidal and striatonigral projections: two distinct fiber systems in primate. Brain Res. 303, 385–390. Parent A. and Hazrati L. N. (1993) Common structural organization of two output nuclei of primate basal ganglia. Trends Neurosci. 16, 308–309. Parent A. and Hazrati L. N. (1995) Functional anatomy of basal ganglia. I. The cortico-basal ganglia-thalamo-cortical loop. Brain Res. Rev. 20, 91–127. Pellegrino L. J. and Cushman A. J. (1967) A Stereotaxic Atlas of the Rat Brain. Appleton-Century-Crofts, New York. Rupniak N. M. J., Jenner P. and Marsden C. D. (1983) Cholinergic modulation of perioral behavior induced by chronic neuroleptic administration to rats. Psychopharmacology 79, 226–230. Rupniak N. M. J., Jenner P. and Marsden C. D. (1986) Acute dystonia induced by neuroleptic drugs. Psychopharmacology 88, 403–419. Salamone J. D. and Baskin P. B. (1996) Vacuous jaw movements induced by reserpine and low-dose apomorphine: possible model of parkinsonian tremor. Pharmac. Biochem. Behav. 53, 179–183. Salamone J. D., Lalies M. D., Channell S. L. and Iversen S. D. (1986) Behavioral and pharmacological characterization of the mouth movements induced by muscarinic agonists in the rat. Psychopharmacology 88, 467–471. Salamone J. D., Johnson C. J., McCullough L. D. and Steinpreis R. E. (1990) Lateral striatal cholinergic mechanisms involved in oral motor activities in the rat. Psychopharmacology 102, 529–534. Steinpreis R. E., Baskin P. P. and Salamone J. D. (1993) Vacuous jaw movements induced by sub-chronic administration of haloperidol: interactions with scopolamine. Psychopharmacology 111, 99–105. Steinpreis R. E. and Salamone J. D. (1993) The effects of acute haloperidol and reserpine administration on vacuous jaw movements in three different age groups of rats. Pharmac. Biochem. Behav. 46, 405–409. Stewart B. R., Jenner P. and Marsden C. D. (1988) The pharmacological characterization of pilocarpine-induced chewing in the rat. Psychopharmacology 97, 228–234. Stewart B. R., Jenner P. and Marsden C. D. (1989) Assessment of the muscarinic receptor subtype involved in the mediation of pilocarpine-induced purposeless chewing behaviour. Psychopharmacology 97, 228–234. Tarsy D. (1983) Neuroleptic-induced extrapyramidal reactions: classification, description and diagnosis. Clin. Neuropharmac. 6, s9–s26. Trevitt J., Lyons M., Aberman J., Carriero D., Finn M. and Salamone J. D. (1997) Effects of clozapine, thioridazine and haloperidol on behavioral tests related to extrapyramidal motor function. Psychopharmacology (in press). von Krosigk M., Smith Y., Bolam J. P. and Smith A. D. (1992) Synaptic organization of GABAergic inputs from the striatum and the globus pallidus onto neurons in the substantia nigra and retrorubral field which project to the medullary reticular formation. Neuroscience 50, 531–549.
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Wickens A. P. and Pertwee R. G. (1995) Effect of delta 9-tetrahydrocannabinoid on circling in rats induced by intranigral muscimol administration. Eur. J. Pharmac. 282, 251–254. 54. Wirshing W. C., Cummings J. L., Dencker S. J. and May P. R. (1989) Electromechanical characteristics of tardive dyskinesia. Schizophr. Res. 2, 40. 55. Young A. B. and Penney J. B. (1993) Biochemical and functional organization of the basal ganglia. In Parkinson’s Disease and Movement Disorders (eds Jankonvic J. and Tolosa E.), pp. 1–12. Williams and Wilkins, Baltimore. (Accepted 12 February 1997)
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Neuroscience Vol. 80, No. 2, pp. 535–544, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(97)00087-0
INVOLVEMENT OF PALLIDAL AND NIGRAL GABA MECHANISMS IN THE GENERATION OF TREMULOUS JAW MOVEMENTS IN RATS M. FINN, A. J. MAYORGA, A. CONLAN and J. D. SALAMONE* Department of Psychology, University of Connecticut, Storrs, CT 06269-1020, U.S.A. Abstract––Four experiments were conducted to investigate the role of pallidal and nigral GABA in the generation of tremulous jaw movements in rats. In these experiments, tremulous jaw movements were induced by i.p. injections of the anticholinesterase tacrine (5.0 mg/kg). Previous work has shown that the tremulous jaw movements induced by cholinomimetics and dopamine depletion are dependent upon striatal mechanisms. Thus, the present study investigated potential striatal output pathways that could be involved in the generation of these movements. Because there are GABAergic projections from neostriatum to entopeduncular nucleus (medial globus pallidus) and substantia nigra pars reticulata, the GABA agonist muscimol was injected directly into these structures to study the effects of GABA stimulation on tacrine-induced jaw movements. Injections of muscimol into the entopeduncular nucleus (25–100 ng) failed to have any significant effects on tacrine-induced vacuous jaw movements. However, injections of muscimol (12.5–50 ng) into the substantia nigra pars reticulata blocked the jaw movements induced by tacrine. In the third experiment, it was again demonstrated that 25.0 ng of muscimol injected directly into the substantia nigra pars reticulata blocked the jaw movements induced by tacrine; in addition, it was shown that injections of this dose 2.0 mm dorsal to the substantia nigra pars reticulata failed to affect tacrine-induced tremulous jaw movements. It was shown in the fourth experiment that injections of muscimol into a more medial portion of the substantia nigra pars reticulata also reduced tacrine-induced tremulous jaw movements. These results indicate that stimulation of GABAA receptors in substantia nigra pars reticulata can block tacrine-induced tremulous jaw movements. This finding is consistent with the notion that striatonigral GABA projections are involved in the generation of tremulous jaw movements. It is also possible that striatonigral GABA mechanisms are involved in human clinical phenomena such as parkinsonian tremor. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: acetylcholine, tremor, Parkinsonism, substantia nigra, globus pallidus, motor, striatum, dopamine.
Drug-induced perioral movements in rats have frequently been used to study extrapyramidal motor function.15,41,43 One of the most widely investigated of these perioral movements is vacuous or tremulous jaw movements (also known as vacuous or purposeless chewing). Vacuous jaw movements are defined as vertical deflections of the lower jaw that resemble chewing but are not directed towards any particular stimuli (e.g., Refs 44 and 45). These movements are induced by dopamine antagonists,41,46,47 reserpine,4,43,47 and neurotoxic depletions of dopamine in the ventrolateral portion of the neostriatum.18,27 Stimulation of muscarinic acetylcholine receptors also leads to vacuous jaw movements.5,9,41,44,45,49 As was the case with dopamine depletions, striatal mechanisms appear to be critical for the generation of cholinomimetic-induced vacuous jaw movements. Kelley et al.29 injected physostigmine into several striatal subregions, and reported that the most effective site was the ventrolateral striatum. Salamone *To whom correspondence should be addressed. Abbreviations: SNr, substantia nigra pars reticulata.
et al.45 observed that the ventrolateral striatum was the most effective site for the induction of jaw movements by the muscarinic agonist pilocarpine. In a recent study,34 it was reported that the anticholinesterase tacrine induced vacuous jaw movements in the range of 2.5–10.0 mg/kg. The jaw movements induced by 5.0 mg/kg tacrine were completely blocked by injection of 2.5–10 µg scopolamine into the ventrolateral striatum.34 Although the clinical significance of vacuous jaw movements remains uncertain (see Refs 15 and 42), it has been suggested that vacuous jaw movements could represent a rat model of parkinsonian tremor.18,27,34,45 Using a slow-motion videotape method, it has been shown that the jaw movements induced by reserpine,43 striatal dopamine depletion,18 pilocarpine18 and tacrine34 occur as repetitive bursts of movement with a peak in the 3.0–6.6 Hz frequency range. Electromyographic analyses of jaw muscles indicate that the temporalis muscle shows rhythmic bursts of activity that correspond to the tacrine-induced jaw movements.11 The local frequency of the jaw movements induced by dopamine
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depletion and cholinomimetics is consistent with the parkinsonian resting tremor frequency,1,26 but is not consistent with the 1–2 Hz frequency typically reported for tardive dyskinesia.54 Thus, it has been noted that these jaw movements are not only vacuous (i.e. non-directed), but are also tremulous (i.e. characterized by a periodic oscillation of a body member, see Refs 16 and 17). The acetylcholine–dopamine interaction that occurs in tremulous jaw movements is similar to that shown in human Parkinsonism. For example, tremulous jaw movements are induced by striatal dopamine depletions and cholinomimetic drugs; these conditions are also associated with human Parkinsonism.3,14,23,35,36 Tremulous jaw movements induced by reserpine can be reduced by the anticholinergic scopolamine,43 and it has been shown that anticholinergic drugs reduce parkinsonian symptoms but exacerbate tardive dyskinesia.7,14 The jaw movements induced by pilocarpine or reserpine can be reduced by moderate doses of the dopamine agonist apomorphine,4,48 and more recently it was shown that the jaw movements induced by the anticholinesterase tacrine can be reduced by various antiparkinsonian drugs, including apomorphine, bromocriptine, amantadine, L-DOPA and benztropine.12 Thus, it is possible that further research into tremulous jaw movements could be useful for helping to characterize the neurochemical and anatomical basis of extrapyramidal motor functions, particularly those mechanisms related to Parkinsonism. One of the most important questions remaining in this area concerns the anatomical pathway that is necessary for generating tremulous jaw movements. As noted above, striatal dopamine and acetylcholine mechanisms are critical for the generation of these movements. Yet the striatal output pathway through which jaw movements are stimulated remains uncertain. The two major striatofugal pathways project either to the medial segment of the globus pallidus (or entopeduncular nucleus in non-primates) or the substantia nigra pars reticulata (SNr).19,20,32,37,38 The medial globus pallidus and SNr are both considered to be major output nuclei of the basal ganglia.39,55 The direct striatofugal pathways utilize GABA as a neurotransmitter,21,52,55 and stimulation of GABA receptors in the SNr has been shown to produce motor effects such as circling behaviour.53 Thus, it is possible that manipulation of GABA transmission in the entopeduncular nucleus or SNr could affect the neural circuitry that generates tremulous jaw movements. Four experiments were conducted to study the role of pallidal and nigral GABA in the production of tremulous jaw movements. In the first two experiments, microinjections of the GABAA agonist muscimol into the entopeduncular nucleus and the lateral SNr of the rat were studied for their effects upon tacrine-induced tremulous jaw movements. Based upon the results of the first two experiments, a third experiment was conducted in which the effects of
intra-nigral muscimol were replicated, and muscimol was also injected into a site dorsal to the SNr in order to control for possible spread of the drug from the SNr injection site. In the fourth experiment, muscimol was injected into a more medial SNr injection site. EXPERIMENTAL PROCEDURES
Subjects The subjects were 113 male Sprague–Dawley rats obtained from Harlan Sprague–Dawley (Indianapolis, IN). All rats were housed individually in a colony room maintained at 23)C with a 12 h light/dark cycle (lights on at 07.00). Standard lab chow and water were available ad libitum and all behavioural testing was performed 4–9 h after light onset. University guidelines on animal care and experimentation were followed for these studies. Drugs All drugs were obtained from Sigma Chemical Co. Both tacrine and muscimol were dissolved in 0.9% saline. Tacrine was administered i.p. immediately following the intracranial injection of muscimol or saline. Behavioural testing was initiated 10 min after the injection of tacrine. Behavioural observation procedures The observation chamber consisted of a clear Plexiglas box measuring 28#28#28 cm3, which had a wire mesh floor. The box was elevated 42 cm from the surface of the table, allowing behavioural observation from all angles. An observer who was unaware of the drug treatment recorded vacuous jaw movements for a 5 min period using a mechanical counter. Vacuous jaw movements were defined as vertical deflections of the lower jaw that resembled chewing but were not directed at any particular stimulus. Yawning, gaping and tongue protrusions were not counted as vacuous jaw movements. Each individual deflection of the jaw was counted as a jaw movement. Previous research has shown a high degree of interrater reliability (i.e. correlations between two observers greater than r=+0.9) with the use of these observational methods for recording jaw movements (see Ref. 34). Surgery Rats were anaesthetized with sodium pentobarbital (50.0 mg/kg). Stainless steel guide cannulae were implanted using standard stereotaxic procedures. The stereotaxic coordinates for the entopeduncular nucleus were the following (in mm, with incisor bar 5.0 mm above interaural line, see Ref. 40): A-P, "0.6 from bregma, M-L, &2.7 from midline, D-V, "6.4 from skull. For the lateral SNr site, the coordinates were A-P, "3.0, M-L, &2.8, D-V, "7.0. The coordinates for the dorsal control site were A-P, "3.0, M-L, &2.8, D-V, "5.0. The coordinates for the middle SNr site were: A-P, "3.0, M-L, &1.8, D-V, "7.2. The cannulae were anchored to the skull with stainless steel screws and dental cement. Wire stylets were placed into the guides to prevent occlusion. A recovery period of seven days was allowed before experimental testing. Intracranial drug injections Injections were made with 30-gauge injection cannulae extending 2.0 mm below the guide cannulae for the SNr (D-V, "9.0 from the skull for lateral SNr, D-V, "9.2 for middle SNr) and the dorsal control site (D-V, "7.0), and 1.0 mm below the guide cannulae for the entopeduncular nucleus (D-V, "7.4). The injectors were attached via PE-10 tubing to a 10 µl Hamilton syringe driven by a syringe pump
Nigral GABA and jaw movements (Harvard Apparatus). Bilateral microinfusions were made in a total volume of 1.0 µl, at a rate of 0.5 µl/min for each side. The injection cannulae were left in place for 1 min following drug infusion to allow for diffusion.
Table 1. The effect of muscimol injected into entopeduncular nucleus on tacrine-induced tremulous jaw movements in experiment 1 Drug treatment
Experiments In the first experiment rats were injected with either saline (n=7), 25.0 (n=7), 50.0 (n=6) or 100.0 ng (n=6) of muscimol per side into the entopeduncular nucleus, followed by 5.0 mg/kg of tacrine i.p. In the second experiment, 29 rats received intracranial injections of either saline (n=8), 12.5 (n=7), 25.0 (n=7) or 50.0 ng (n=7) of muscimol per side into the lateral SNr, followed by 5.0 mg/kg of tacrine i.p. In the third experiment, three groups were compared. One group (n=8) had cannulae implanted into lateral SNr, as described above, and received injections of 1.0 ul saline vehicle through the cannulae as well as 5.0 mg/kg tacrine. A second group (n=9) also had cannulae implanted into lateral SNr, and received tacrine plus 25.0 ng muscimol per side. In the third group, rats (n=12) were implanted with an injection target site 2.0 mm dorsal to the SNr (dorsal control site, see above). These rats received 25.0 ng of muscimol per side through the cannulae followed by 5.0 mg/kg of tacrine i.p. In the fourth experiment, rats were implanted with cannulae in the middle SNr as described above, and received intracranial injections of saline (n=8), 6.25 (n=6), 12.5 (n=7) or 25.0 ng (n=8) of muscimol per side, followed by 5.0 mg/kg of tacrine i.p. All animals were observed as described above for a 5 min period that was initiated 10 min after the tacrine injection. Histology Upon completion of the experiment, rats were deeply anaesthetized with sodium pentobarbital and perfused transcardially with physiological saline followed by 10% formalin to fix the brains. The brains were then removed and placed in 10% formalin for at least two days. They were cut into 75 µm coronal sections, mounted on glass slides and stained with Cresyl Violet to determine the location of the cannulae tracks. Data analysis Jaw movement data in each experiment were analysed by simple analysis of variance (ANOVA; Systat 5.0). Each dose of muscimol was compared with the effects of saline vehicle injection by using non-orthogonal planned comparisons that employed the overall error term from the ANOVA.30 RESULTS
Table 1 shows the results of the first experiment, in which muscimol was injected into the entopeduncular nucleus. There was no significant effect of muscimol on the jaw movements induced by 5.0 mg/kg tacrine (F3,22=0.637, P=0.599). The effects of SNr injections of muscimol on tremulous jaw movements induced by 5.0 mg/kg tacrine (the second experiment) are shown in Table 2. As shown in this table, there was a significant reduction in tremulous jaw movements produced by injection of muscimol (F3,25=5.966, P<0.01). Planned comparisons indicated that 25.0 ng and 50.0 ng doses of muscimol significantly reduced tremulous jaw movements relative to tacrine alone. In Table 3, the results of the third experiment are shown. There was an overall effect of drug treatment on tremulous jaw movements (F2,26=5.95, P<0.01). Planned comparisons revealed that injections of 25.0 ng muscimol into the SNr significantly reduced
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5.0 mg/kg tacrine+vehicle 5.0 mg/kg tacrine+25.0 ng/ side muscimol 5.0 mg/kg tacrine+50.0 ng/ side muscimol 5.0 mg/kg tacrine+100 ng/ side muscimol
Number of jaw movements Mean 168.0
S.E.M. 25.7
135.3
23.0
112.3
34.5
131.0
34.0
All rats received 5.0 mg/kg tacrine, as well as injections of vehicle or muscimol into the entopeduncular nucleus. Mean (&S.E.M.) number of jaw movements per 5 min observation period is shown for each treatment condition. Table 2. The effect of muscimol injected into substantia nigra pars reticulata on tacrine-induced tremulous jaw movements in experiment 2 Drug treatment 5.0 mg/kg tacrine+vehicle 5.0 mg/kg tacrine+12.5 ng/ side muscimol 5.0 mg/kg tacrine+25.0 ng/ side muscimol 5.0 mg/kg tacrine+50.0 ng/ side muscimol
Number of jaw movements Mean 125.1
S.E.M. 24.5
79.1
21.4
26.0*
13.4
29.1*
14.0
*P<0.05, different from tacrine plus vehicle. All rats received 5.0 mg/kg tacrine, as well as injections of vehicle or muscimol into the substantia nigra pars reticulata. Mean (&S.E.M.) number of jaw movements per 5 min observation period is shown for each treatment condition. Table 3. The results of experiment 3 Drug treatment
Mean
S.E.M.
5.0 mg/kg tacrine+vehicle/SNr 5.0 mg/kg tacrine+25.0 ng muscimol/SNr 5.0 mg/kg tacrine+25.0 ng muscimol/dorsal
102.8
19.9
33.0* 122.8
10.6 17.3
*P<0.05, different from tacrine plus vehicle. All rats received 5.0 mg/kg tacrine, as well as injections of either vehicle into substantia nigra pars reticulata (SNr), 25.0 ng muscimol into the SNr or 25.0 ng muscimol into the dorsal control site. Mean (&S.E.M.) number of jaw movements per 5 min observation period is shown for each treatment condition.
tremulous jaw movements relative to tacrine alone; this finding replicates the results of the previous study. However, injections of 25.0 ng muscimol 2.0 mm dorsal to SNr into overlying brain areas had no significant effect on tremulous jaw movements. The effects of muscimol injected into the middle SNr are shown in Table 4. There was a significant overall
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Table 4. The effect of muscimol injected into the middle substantia nigra pars reticulata site on tacrine-induced tremulous jaw movements in experiment 4 Drug treatment
Number of jaw movements
5.0 mg/kg tacrine+vehicle 5.0 mg/kg tacrine+6.25 ng/ side muscimol 5.0 mg/kg tacrine+12.5 ng/ side muscimol 5.0 mg/kg tacrine+25.0 ng/ side muscimol
Mean 87.8
S.E.M. 16.9
17.3*
9.3
11.4*
7.3
2.8*
1.8
*P<0.05, different from tacrine plus vehicle. All rats received 5.0 mg/kg tacrine, as well as injections of vehicle or muscimol into the substantia nigra pars reticulata. Mean (&S.E.M.) number of jaw movements per 5 min observation period is shown for each treatment condition.
effect of muscimol on tacrine-induced tremulous jaw movements (F3,25=13.2, P<0.001). Planned comparisons revealed that all three doses of muscimol plus tacrine significantly differed from tacrine alone. All cannula placements were verified by histological analyses. For simplicity, only those animals that received 25.0 ng muscimol in each of the four experiments have their placements shown in Figs 1–3 (left hemisphere only). Analysis of the histology demonstrated that cannulae were placed in the entopeduncular nucleus in the first experiment (Fig. 1), and in the anterolateral SNr in the second experiment (Fig. 2). The results of the histological analyses of the third and fourth experiments are shown in Fig. 3. For the group of rats that received injections of muscimol into the SNr, it can be seen that the placements are in the vicinity of the anterolateral SNr, as in the previous experiment. Rats in the dorsal control group had placements in the area of the zona incerta, posterior thalamus, lateral geniculate and reticular formation. One of the placements in this group was near the substantia nigra pars compacta, but none of them were in the subthalamic nucleus. The middle SNr placement (experiment 4) resulted in injections sites that were centrally located within the anterior SNr region. DISCUSSION
Stimulation of GABAA receptors in SNr by local injections of muscimol produced a dose-related reduction in tacrine-induced tremulous jaw movements. With placements in the lateral SNr, this effect was significant with injections of 25.0 ng muscimol, and some animals that received 25.0 or 50.0 ng muscimol showed a complete blockade of tacrineinduced tremulous jaw movements. The middle SNr injection site resulted in a more potent effect of muscimol, with significant effects at 6.25 and 12.5 ng doses. The effects of muscimol on tremulous jaw
movements were anatomically specific. There were no significant effects of muscimol injections into entopeduncular nucleus, even if doses as high as 100.0 ng were used. Moreover, injections of 25.0 ng muscimol 2.0 mm dorsal to SNr also failed to reduce tremulous jaw movements. The latter results suggest that muscimol injected into SNr is not reducing tremulous jaw movements simply because the drug is diffusing along the guide cannulae to other, more distant brain regions. Although the substantia nigra pars compacta is immediately dorsal to the SNr, it seems unlikely that muscimol is acting in this region to reduce tremulous jaw movements. GABAergic inhibition of substantia nigra dopamine-containing cells should, if anything, increase tremulous jaw movements. Previous work has shown that tremulous jaw movements are induced by dopamine antagonism or striatal dopamine depletion.4,18,27,46,47 Injections of low, presynaptically active doses of apomorphine increase, rather than decrease, reserpine-induced jaw movements.4 Tacrine-induced jaw movements are not blocked by doses of haloperidol up to 1.0 mg/ kg,51 so it is unlikely that inhibition of dopamine cells would block these movements. In addition, some of the rats that received implantations targeted at the SNr had placements that were quite ventral to the substantia nigra pars compacta; these rats still showed a blockade of tacrine-induced jaw movements. Thus, the present evidence, as well as previous work, suggests that stimulation of GABAA receptors in SNr reduces tremulous jaw movements. Anatomical evidence indicates that the medial globus pallidus/ entopeduncular nucleus and SNr are the two major output nuclei for both the ‘‘direct’’ and ‘‘indirect’’ pathways of striatal output.2,13,39 The former pathway represents a direct GABAergic projection from medium spiny cells in the neostriatum to the globus pallidus or SNr, while the latter involves connections through lateral globus pallidus and subthalamic nucleus. The present study demonstrates that the entopeduncular nucleus and SNr in the rat are not functionally equivalent in terms of tremulous jaw movements (see also Ref. 6). Rather, tremulous jaw movements seem to be generated through a mechanism that involves SNr, but not entopeduncular nucleus. Given current models of basal ganglia function, it is not clear why large doses of muscimol in the entopeduncular nucleus are ineffective for the suppression of tremulous jaw movements. It is possible that there are species differences in terms of the relative importance of medial globus pallidus and SNr as striatal output areas. Also, it should be mentioned that parkinsonian tremor in primates usually involves the hand; although jaw tremors do occur,1,25,50 they are less common than hand tremors. In rats, although we occasionally observe paw tremors, it is the jaw movements that are most obvious after dopamine depletions and cholinergic stimulation. It is possible that there are differences between the pathways through which forelimb and orofacial
Nigral GABA and jaw movements
Fig. 1. Cannulae placements for the first experiment are shown here. Placements are depicted for rats that received injections of 25.0 ng muscimol into the entopeduncular nucleus (black circles). EP, entopeduncular nucleus; see also Ref. 40.
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Fig. 2. Cannulae placements for the second experiment are shown here. Placements are shown for rats that received injections of 25.0 ng muscimol into the substantia nigra pars reticulata (black squares). SN, substantia nigra; see also Ref. 40.
Nigral GABA and jaw movements
Fig. 3. Placements for the third and fourth experiments are shown in this figure; placements are depicted for rats that received injections of 25.0 ng muscimol into the SNr (large black squares) and the dorsal control site (black circles) in experiment 3, as well as placements in the middle SNr site (triangles) in experiment 4. SN, substantia nigra; ZI, zona incerta; GL, lateral geniculate; see also Ref. 40.
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movements are influenced by striatal output. In rats, and possibly in other species as well, the SNr may be more important than the medial globus pallidus/ entopeduncular nucleus for conveying information that is necessary for oral motor function. It has been demonstrated that circuits connecting neostriatum, substantia nigra and superior colliculus are important for the generation of eye movements;10,28 future research will be necessary to determine the pathway through which cholinomimetic-stimulated jaw movements are generated. CONCLUSIONS
It has been suggested that neurons in pallidal output regions are hyperactive under the conditions that lead to Parkinsonism, and there is evidence to support this contention from parkinsonian patients.26 In fact, the presence of pallidal hyperactivity has been used as a rationale for using pallidotomies as a treatment for Parkinsonism.26,32 In the present study, it was observed that muscimol injected into SNr was very potent at reversing cholinomimetic-induced tremulous movements. This finding is consistent with the notion that neurons in the SNr are hyperactive during tremulous jaw movements, and that GABA-induced inhibition of SNr reverses both the neuronal hyperactivity and the jaw movements. The precise reason why conditions that lead to Parkinsonism would cause hyperactivity in
SNr neurons is not certain, although models of basal ganglia function have been proposed to explain this type of effect (e.g., Refs. 13 and 55). It is possible that interference with dopamine or stimulation of acetylcholine in the striatum leads to an inhibition of GABAergic neurons that project to SNr via the ‘‘direct’’ pathway.55 Also, it is possible that, through the connections with the lateral globus pallidus and subthalamic nucleus, parkinsonian conditions lead to an excess of glutamate-mediated excitation of SNr.6,22,31 Future research will be necessary to determine if either decreased GABA release or increased glutamate release in SNr is associated with the production of tremulous jaw movements in rats. Additional studies will be undertaken to identify other pharmacological conditions (e.g., SNr injections of GABA antagonists, excitatory amino acid agonists) that could lead to the production of tremulous jaw movements. Whether it is through glutamate or GABA mechanisms, or some combination of the two, the hyperexcitability of cells in the SNr could be a critical trigger for the generation of tremulous motor activity. Through connections with the thalamus or brainstem motor areas,52 the SNr could be instigating the oscillatory neural activity that ultimately manifests itself as tremulous movements.8 Acknowledgements—Many thanks to Bill Lexton, for his assistance with animal care. This work was supported by a grant from NIH (NINDS).
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